Mass spectrometer with a laser desorption ion source, and laser system with a long service life

ABSTRACT

The invention relates to a mass spectrometer with laser-desorption ion source, particularly for MALDI. A laser system with optical laser spot control is proposed in which the laser spot shift brought about by means of a temporally variable angular deflection at a mirror system is performed on the laser beam before or during the energy multiplication. The laser beam, which is deflected through a small angle by the mirror system, is converted by a suitable flat-field optical system into a parallel-shifted laser beam, which then passes through a multiplier crystal. After exiting the multiplier crystal system, the parallel-shifted beam is converted back into a slightly angled beam by a flat-field optical system, this latter beam then bringing about the spot shift on the sample. The multiplier crystal is conserved by the continuously temporally changed parallel shift of the laser beam in the multiplier crystal, thus prolonging its service life.

The invention relates to a mass spectrometer with laser desorption ionsource, which ionizes the analyte molecules of a sample by means oflaser desorption from a sample support, in particular by means ofmatrix-assisted laser desorption. The associated laser system has anextended service life.

PRIOR ART

Over the past twenty years, two types of ionization have becomeestablished in the mass spectrometry of biological macromolecules:ionization by matrix-assisted laser desorption (MALDI), and electrosprayionization (ESI). The biological macromolecules to be analyzed aretermed analyte molecules below. In the MALDI method, the analytemolecules are generally prepared on the surface of a sample support, ina solid, polycrystalline matrix layer, and are predominantly singlyionized, whereas in the ESI method they are dissolved in a liquid andmultiply ionized. It was these two methods which first made possible themass-spectrometric analysis of biological macromolecules forinvestigations in genomics, proteomics and metabolomics; theirinventors, John B. Fenn and Koichi Tanaka, were awarded the Nobel Prizein Chemistry in 2002.

Matrix-assisted laser desorption and ionization has been sustainablyimproved in recent years by switching from nitrogen lasers to solidstate UV lasers with a longer service life, and in particular by usingbeam generation with a spatially modulated beam profile for an increasedion yield. The method of beam generation and the corresponding lasersystems have been described in the equivalent documents DE 10 2004 044196 A1, GB 2 421 352 B and U.S. Pat. No. 7,235,781 B2 (A. Haase et al.,2004) and are known by the name “smartbeam”.

The “smartbeam” is based on the finding that the ionization rate of thesample material increases greatly when the laser spots are made verysmall, down to a few micrometers. Only relatively few ions are formed inthese small laser spots, however, and it is therefore expedient togenerate several spots simultaneously, i.e. to use a pattern of laserspots.

In time-of-flight mass spectrometers with ionization of the samples bymatrix-assisted laser desorption (MALDI), the laser beam is usuallyfocused, by means of lenses and mirrors in fixed adjustment, onto asample on a sample support such that the laser spot with the desireddiameter and energy density (or a pattern of such laser spots) impingesin the ion source at a location on the sample support which is optimallyspecified for high sensitivity. The samples on the sample supportcontain a thin layer of tiny crystals of the matrix substance, in eachof which a small quantity of analyte molecules is embedded. The samplesare each moved into the focus of the laser spot by a mechanical movementof the sample support. A light pulse from the laser, usually a pulsed UVlaser, produces a plasma cloud of the sample material, in which ions ofthe matrix and analyte molecules are produced.

Modern embodiments of MALDI lasers (see DE 10 2004 044 196 A1; A. Haaseet al., 2004, corresponding to GB 2 421 352 B; U.S. Pat. No. 7,235,781B2) produce not just a single irradiation spot, but a pattern of severalirradiation spots simultaneously, whereby spot diameter and energydensity can be optimized in such a way that the achieved yield ofanalyte ions can be ten to one hundred times higher. The pattern cancontain 4, 9 or 16 irradiation spots in a square arrangement, forexample, but also 7 or 19 spots in a hexagonal arrangement. The moreeconomic use of the sample material allows the utilization factor of thesamples to be increased.

The publication DE 10 2013 018469 A1 (A. Haase; corresponding to GB 2521 730 A and US 2015/0122986 A1) elucidates a very simple and low-costmethod to generate spot patterns with five or nine spots in a squarearrangement.

The generation of patterns increases the ion yield per analyte moleculeby far more than a factor of 10 and reduces the sample consumptionaccordingly; this is important especially for imaging mass spectrometryon thin tissue sections. Since modern mass spectrometers are designedfor a spectral acquisition repetition rate of 10,000 spectra per secondand more, the energy efficiency of the generation of the spot pattern isvery important if the aim is to avoid the need for expensivehigh-performance lasers.

The patent specification DE 10 2011 112 649 B4 (A. Haase et al.; seealso U.S. Pat. No. 8,872,102 B2 and GB 2 495 805 A) presents a lasersystem for MALDI applications which allows an optical movement of thelaser spot (or laser spot pattern) on the sample and thus facilitatesfaster, lower-inertia scanning of a sample than can be achieved with amovement of the mechanically slow sample support plate. The temporallyvariable spot control is based on two small, low-inertia galvanometermirrors, which deflect the UV beam to a position where the diameter ofthe UV beam is of the order of millimeters. The beam here is deflectedthrough tiny angles in two spatial directions; the deflection is thentranslated into a movement of the tiny laser spot or laser spot patternin a square area with an edge length of a few 100 micrometers by meansof a telescope and a large-diameter lens. The fast galvanometer mirrorscan sometimes move the spots in less than 100 microseconds, whichcorresponds well with the desired laser pulse frequency of 10,000 lasershots per second. The cited patent specification DE 10 2011 112 649 B4and all its content shall be included here by reference. The principleof temporally variable laser spot control is shown in FIG. 1.

The laser system described in patent specification DE 10 2011 112 649 B4has a service life of several billion laser shots. For continuousoperation at full shot capacity, this corresponds to a service life ofaround 35 days; for eight hours of operation on five days a week, theservice life is around 150 days. This service life still leaves room forimprovement. There is a need for a laser system which has a longerservice life, for example ten times that.

Investigations have shown that the service life is limited by adegradation of the multiplier crystals. The beam exit site of thetripling crystal suffers in particular, because here the UV beamdecomposes unavoidable deposits of tiny particles or organic moleculeson the surface and thus creates a site where the absorption continuallyincreases. It is fundamentally not possible to completely remove theorganic substances occurring only in minute trace concentrations. Thereeven appear to be mechanisms which cause the tiny particles and organicmolecules to migrate to the surface of the tripling crystal.

It is known that the service life of this tripling crystal can beincreased by parallel shifting of the crystal so that the UV beam, whichhas a diameter of only a few hundred micrometers here, always exits thesurface at fresh locations. This parallel shift fundamentally requiresextremely high precision, since even the slightest tilt drasticallyreduces the nonlinear effect of the crystal. Such a shifting mechanismis complex and expensive; moreover, the electromechanical drive elementscontribute to the contamination of the surrounding gas through wear andtear, for example.

The patent specification U.S. Pat. No. 7,460,569 B2 (Van Saarlos; 2008)describes how a parallel offset of a laser beam can be generated by aplane-parallel plate tilted in relation to the axis and rotated aboutthe beam. The rotation causes the beam to execute a circular movementthrough the multiplier crystals, which increases the service life. Asecond rotatable, plane-parallel plate shifts the beam back into theoriginal axis.

The patent specification U.S. Pat. No. 8,885,246 B2 (D. Horain et al.,2009) describes how an additional tilting movement of the plane-parallelplates can bring about not only a circular parallel movement of thelaser beam, but also a scan which utilizes the entire surface of thecrystals. It also describes how the second plate has to be tiltedindependently of the first in order to bring the beam back accuratelyinto the original axis because the refractive index changes with thebeam wavelength.

The arrangements described in both patent specifications again requireelectromechanical drives that operate very precisely, which can in turncontaminate the surrounding gas. There is therefore still a need formethods and devices to prolong the service life of the laser system formass spectrometers with laser desorption ion sources, especially MALDIsources.

SUMMARY OF THE INVENTION

For a mass spectrometer with laser desorption ion source, a laser systemwith temporally variable optical laser spot control is proposed in whichthe laser spot shift, which is produced by means of an angulardeflection in a mirror system (for example at two galvanometer mirrors),is performed not on the frequency-increased laser beam after it has leftthe multiplier crystal system, but on the laser beam before or duringthe multiplication of the energy. The beam, which is deflected through asmall angle by the mirrors, is converted by a high-quality flat-fieldoptical system into a parallel-shifted laser beam, which then passesthrough a multiplier crystal (for example a doubling crystal and atripling crystal). After exiting the multiplier crystal system, theparallel-shifted beam is converted back into a slightly angled beam by asecond flat-field optical system, and the beam then brings about thespot shift on the sample in the way which is already known. Uniformscanning of the sample therefore leads automatically to uniform scanningof the multiplier crystals. The parallel shift of the laser beam infront of or within the multiplier crystal system causes the exit site ofthe frequency-increased laser beam from the multiplier crystal to shiftalong with the spot control on the sample in the desired way, thusincreasing the service life.

The invention therefore relates to a mass spectrometer (for example forimaging mass spectrometry) which has an ion source which ionizes sampleson a sample support by means of laser desorption (for example via MALDI)and contains a laser system for this purpose and has a mass analyzer todetect the ions produced. The laser system comprises the followingsubsystems: a) a laser (for example IR laser) to generate laser beampulses of long-wavelength light, b) a multiplier crystal system togenerate laser beam pulses of short-wavelength light from the laser beampulses of long-wavelength light, c) a mirror system for the positioncontrol of laser spots on the sample support by means of an angulardeflection of laser beam pulses, d) a first flat-field optical systembehind the mirror system to transform the angular deflection of thelaser beam pulses into a parallel shift (for example parallel to an axisof the multiplier crystal system), and e) a second flat-field opticalsystem behind the multiplier crystal system to transform the parallelshift back into an angular deflection of the laser beam pulses ofshort-wavelength light.

The mirror system preferably contains two movable mirrors to deflect thelaser spots in both spatial directions at right angles to the surface ofthe sample support, for example one or two galvanometer mirrors. Allelements which deflect light by means of reflection shall befundamentally considered as a mirror in the context of this disclosure.

Different embodiments may additionally have a pattern generator togenerate a laser spot pattern on the surface of the sample support.

The mass analyzer is preferably a time-of-flight analyzer with axial ororthogonal ion injection (TOF-MS), an ion-cyclotron-resonance analyzer(ICR-MS), a radio frequency voltage ion trap (IT-MS) or an electrostaticion trap of the Kingdon type.

The first flat-field optical system can be positioned in front of themultiplier crystal system. In this case, all subsequent multipliercrystals benefit from the laser beam shift and the associated materialconservation.

In different embodiments, the multiplier crystal system can have two (ormore) multiplier crystals, for example one doubling crystal and onetripling crystal. The first multiplier crystal can produce visible,green coherent light from infrared coherent light, and the secondmultiplier crystal can produce ultraviolet coherent light from thevisible, green coherent light and the infrared coherent light. Incertain embodiments, the mirror system and the first flat-field opticalsystem can be positioned between the multiplier crystals. The servicelife of a tripling crystal in particular can benefit from the continuouslaser beam shifting because the degradation processes in the crystal areamplified, the higher the photon energy.

In different embodiments, a telescope can be provided to expand theangularly deflected laser beam pulses of short-wavelength light.Furthermore, an object lens can be provided to focus the expanded laserbeam in laser spots on the sample support.

The invention also relates to a method for the operation of a massspectrometer with laser desorption ion source, whose laser systemgenerates laser beam pulses of long-wavelength light, from which amultiplier crystal system produces laser beam pulses of short-wavelengthlight, which are then guided to a sample support in order to generateions there by means of laser desorption, said ions being detected in amass analyzer, and a mirror system deflects the laser beam through anangle such that an area on the sample support is scanned or sampled.Downstream of the mirror system, the laser beam pulses pass through afirst flat-field optical system, which converts the angular deflectioninto a parallel shift. Downstream of the multiplier crystal system, thelaser beam pulses of short-wavelength light pass through a secondflat-field optical system, which converts the parallel shift back intoan angular deflection.

BRIEF DESCRIPTION OF THE ILLUSTRATIONS

FIG. 1 shows a greatly simplified schematic of a MALDI time-of-flightmass spectrometer whose arrangement corresponds to the Prior Art.

FIG. 2 again depicts a schematically greatly simplified example of a newarrangement in the region in front of the Keplerian telescope (9). TheIR beam generated in the infrared laser (2), for example at 1064nanometers, is deflected in both spatial directions by the twogalvanometer mirrors (7) and (8) before it enters the multipliercrystals (5) and (6). A high precision flat-field optical system (3)transforms the angularly deflected IR beam into a beam which here runsextremely parallel to the axis of the multiplier crystals (5) and (6),but is shifted more or less parallel, depending on the angle. The UVbeam exiting the tripling crystal (6), third harmonic generation—THG,for example at 355 nanometers, is then transformed back into anangularly deflected beam by a second flat-field optical system (4).Since, generally, the spot control scans the full movement range for thelaser spot on the sample support (13), this arrangement fully exploitsthe volume of the multiplier crystals (5) and (6) and in particular theexit surface from the tripling crystal (6); this increases the servicelife of the laser system (1).

FIG. 3 is an enlargement of the new example arrangement from FIG. 2 infront of the Keplerian telescope (9).

FIG. 4 depicts an alternative arrangement of the multiplier crystals (5,6) relative to the mirror system (7, 8) and the near-field opticalsystems (3, 4).

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a greatly simplified schematic of a MALDI time-of-flightmass spectrometer with a time-of-flight analyzer (21) and a laser system(1) which controls the laser spot position of the light pulse on thesample support plate (13) by means of a mirror system comprising twogalvanometer mirrors (7, 8). The laser pulse is generated in theinfrared laser (2), its energy is doubled in a doubling crystal (5), andat least partially converted into the UV in a tripling crystal (6). Inthe mirror system, the UV beam is deflected in both spatial directionsby two galvanometer mirrors (7) and (8). The deflected laser beam isthen expanded in a Keplerian telescope (9) and shifted parallel as afunction of the angular deflection. The mirror (10) directs the exitinglaser beam into the very center of the object lens (11), with reducedangular deflection. Depending on the angular deflection, the beam passesthrough the object lens (11) centrally, but at slightly differentangles, thus shifting the position of the spot pattern on the samplesupport plate (13). The ions generated in the plasma clouds of the laserspot pattern are accelerated by voltages at the diaphragms (14) and (15)to form an ion beam (18), which passes through the two deflectioncondensers (16) and (17) to correct its trajectory, and is focused ontothe detector (20) by the reflector (19). This arrangement corresponds tothe Prior Art.

It should be noted here that the beam guidance within a Kepleriantelescope (9) is more complex and FIG. 1 does not reproduce it in realterms for reasons of simplicity, although the illustration doescorrectly reproduce the external effect of the telescope (9) on thelaser light beam. In addition, further optical elements, such as lensesto correct the IR beam from the laser (2), which diverges because ofthermal lenses, and to generate a very narrow beam through themultiplier crystals (5, 6), are not shown. These necessary opticalelements are known to the person skilled in the art without any furtherexplanation, however.

For a mass spectrometer with laser desorption ion source, a laser systemis now proposed, with which the steadily progressive damage to the exitsurface of the multiplier system is reduced not by mechanical parallelshifting of one of the crystals at specified intervals of time toprovide fresh exit sites for the incident laser beam, but by continuousoptical parallel shifting of the laser light beam such that the wholeexit surface of the crystal is used. This also reduces the time thelaser dwells on the same spot on the crystal, which largely preventsaccumulative degradation processes at one spot. No plane-parallel plateswith electromechanical drives are used for rotation and tilting,however, but instead a mirror system, consisting of galvanometermirrors, for example, whose deflection properties do not change as afunction of the wavelength (and which is moreover comparatively low inmass and therefore low in inertia).

To this end, and as is shown in FIG. 2 by way of example, the shift ofthe laser spot here, which is produced by an angular deflection at twogalvanometer mirrors (7) and (8), is not applied the UV beam after ithas exited the tripling crystal (6), but to the infrared beam before theenergy multiplication. The IR beam deflected at small angles by thegalvanometer mirrors (7) and (8) is converted by a high-qualityflat-field optical system (3) into an IR beam, which is shifted veryaccurately parallel to the axis of the multiplier crystals (5) and (6),and then passes through the two multiplier crystals (5) and (6),partially tripling its quantum energy in the process. After exiting thetripling crystal (6), a further flat-field optical system (4) convertsthe parallel-shifted beam back into an angled beam, which then bringsabout the spot shift on the sample in the way which is already known.

FIG. 3 shows an enlarged illustration of the principle of the angulardeflection of the IR beam from FIG. 2. It shall, however, be noted herethat it is a simplified schematic to illustrate the basic idea of theinvention. Many details are not shown here. It is, for example,necessary for the IR beam, which diverges because of a thermal lens inthe exit mirror of the laser (2), to be made parallel again by means ofa lens. The flat-field optical system (3) also serves to reduce thelaser beam to a diameter of a few hundred micrometers in order togenerate a sufficiently high photon density in the multiplier crystals.Finally, the UV beam can also pass through a device to generate the spotpattern and through a variable beam attenuator, which can regulate theintensity by several orders of magnitude, but these are also not shownhere for reasons of clarity.

Since, for most applications, the laser spot control on the samplesupport (13) scans or samples the complete available surface of severalhundred micrometers square, the laser spot control on the sample fullyexploits the exit surface of the tripling crystal (6) by virtue of theparallel shift of the narrow infrared beam, with a diameter of severalhundred micrometers, in the tripling crystal, which for example has auseful square cross-section with an edge length of around threemillimeters. The contamination of the surface brought about by thedecomposition of organic molecules is thus reduced by a factor of aroundten to a hundred, extending the service life accordingly.

It should be noted here that an additional, quite simple procedure toextend the service life of a multiplier crystal can consist in enlargingthe crystal and thus the area available for the beam to pass through.The larger the useful crystal volume, the shorter the time for whichrelevant partial volumes of the crystal are subjected to photons duringthe scanning or sampling.

A flat-field optical system is a lens system with particularly goodcorrection. The flat-field optical systems (3) and (4) only need to bewell corrected for the wavelengths used, however, because chromaticaberrations do not play any part. The flat-field optical system (3)therefore only has to be corrected for infrared in the example given,not for a UV beam. If the first flat-field optical system (3) is locatedbetween two multiplier crystals, for example between a doubling crystal(5) and a tripling crystal (6) as indicated in FIG. 4, the wavelength tobe corrected can be in the green visible region (possibly in theinfrared additionally), for example. In both cases, relatively low-costtypes of glass can be used. For the embodiment shown, the secondflat-field optical system (4) must, however, be made fromUV-transmitting material, silica glass, for example, but likewiseoptically corrected for this wavelength only.

Lenses, which are normally required to adjust the diameters of the laserbeam between the crystals (5) and (6), are omitted in FIG. 4 for reasonsof clarity.

The fast scanning of the whole exit surface of the tripling crystal (6)according to the invention is far superior to the mechanical parallelshifting of the multiplier crystals or the use of plane-parallel platesto shift the beam. Mechanical shifting, rotation or tilting is so slowcompared to the utilized pulse rate of the laser that an exit site issubjected to many laser shots in succession at a pulse rate of 10kilohertz. The situation is different for the fast optical scanningproposed. The continuous scanning of the whole area means that eachlaser shot can impinge on a new site of the crystal exit surface, andeach site has a certain time to regenerate until the whole area has beenscanned completely and it is the turn of this site again and the beampasses through it again. For example, the complete scanning or samplingof a square sample area with 0.3-millimeter edge length with a patternof nine laser spots, each with a diameter of five micrometers, requiresmore than 1,000 laser shots; each exit site thus has around 0.1 secondsfor regeneration.

A further important advantage with certain embodiments is the use ofgalvanometer mirrors in the infrared wavelength range, for example at1064 nanometers. The destruction limits here are considerably lower thanthose of the UV wavelength range—by up to a factor of 10. The laser beamat this location can therefore be made smaller than in the UV range.This makes it possible to increase the precision of the position shifton the sample by the galvanometer mirrors by around a factor of three.This means that the deflection system is more tolerant of externalinterferences such as temperature fluctuations.

Different types of mass spectrometer can be used for the invention. Theanalyte ions produced with the laser system can preferably be detectedand analyzed in a special MALDI time-of-flight mass spectrometer withaxial ion injection, as shown schematically in FIGS. 1 and 2. However,it is also possible to feed the analyte ions to other types of massanalyzer for analysis, such as time-of-flight mass spectrometers withorthogonal ion injection (OTOF-MS), ion cyclotron resonance massspectrometers (ICR-MS), radio frequency ion trap mass spectrometers(IT-MS) or electrostatic ion trap mass spectrometers of the Kingdontype.

Multiplier crystal systems with two crystals are described in theexamples shown. It shall, however, be understood that applications whichoperate with a single-step, or more than a two-step, frequency increaseof the photon energy are also conceivable (one crystal, two crystals,three crystals, etc.). The examples must therefore not be seen aslimiting.

Reference was made to MALDI in the explanations above. The principlesshown here can, however, be realized with other laserdesorption/ionization mechanisms without the sample moleculesnecessarily having to be embedded in a matrix. In this respect, thepresent disclosure must be understood in a correspondingly broad way.

Further embodiments of the invention are conceivable in addition to theembodiments described by way of example. With knowledge of thisdisclosure, those skilled in the art can easily design furtheradvantageous embodiments of laser systems for mass spectrometers, whichare to be covered by the scope of protection of the appended claims.

1. A mass spectrometer comprising an ion source which ionizes samples ona sample support by means of laser desorption, and for this purposecomprises a laser system, and a mass analyzer to detect the ionsproduced, where the laser system comprises the following subsystems: a)a laser to generate laser beam pulses of long-wavelength light, b) amultiplier crystal system to generate laser beam pulses ofshort-wavelength light from the laser beam pulses of long-wavelengthlight, c) a mirror system for the position control of laser spots on thesample support by means of an angular deflection of laser beam pulses,d) a first flat-field optical system behind the mirror system totransform the angular deflection of the laser beam pulses into aparallel shift, and e) a second flat-field optical system behind themultiplier crystal system to transform the parallel shift back into anangular deflection of the laser beam pulses of short-wavelength light.2. The mass spectrometer according to claim 1, wherein the mirror systemcontains two movable mirrors to deflect the laser spots in both spatialdirections at right angles to a surface of the sample support.
 3. Themass spectrometer according to claim 2, wherein the mirror systemcontains one or two galvanometer mirrors.
 4. The mass spectrometeraccording to claim 1, further comprising a pattern generator to generatea laser spot pattern on a surface of the sample support.
 5. The massspectrometer according to claim 1, wherein the mass analyzer is one of atime-of-flight analyzer with axial or orthogonal ion injection (TOF-MS),an ion-cyclotron-resonance analyzer (ICR-MS), a radio-frequency voltageion trap (IT-MS) and an electrostatic ion trap of the Kingdon type. 6.The mass spectrometer according to claim 1, wherein the first flat-fieldoptical system is positioned in front of the multiplier crystal system.7. The mass spectrometer according to claim 1, wherein the multipliercrystal system has two multiplier crystals.
 8. The mass spectrometeraccording to claim 7, wherein the first multiplier crystal producesvisible green coherent light from infrared coherent light, and thesecond multiplier crystal produces ultraviolet coherent light from thevisible, green coherent light and the infrared coherent light.
 9. Themass spectrometer according to claim 7, wherein the mirror system andthe first flat-field optical system are positioned between themultiplier crystals.
 10. The mass spectrometer according to claim 1,wherein the laser produces infrared laser light pulses.
 11. The massspectrometer according to claim 1, wherein the ion source operatesaccording to the MALDI principle.
 12. The mass spectrometer according toclaim 1, further comprising a telescope to expand the angularlydeflected laser beam pulses of short-wavelength light.
 13. The massspectrometer according to claim 12, further comprising an object lens tofocus the expanded laser light in laser spots on the sample support. 14.The mass spectrometer according to claim 1, wherein the parallel shiftis executed parallel to an axis of the multiplier crystal system.
 15. Amethod for the operation of a mass spectrometer with laser desorptionion source, whose laser system generates laser beam pulses oflong-wavelength light, which a multiplier crystal system then transformsinto laser beam pulses of short-wavelength light, which are then guidedto a sample support in order to generate ions there by means of la-serdesorption, said ions being detected in a mass analyzer, and a mirrorsystem deflects the laser beam pulses at an angle such that an area onthe sample support is scanned or sampled, wherein the laser beam pulsespass, downstream of the mirror system, through a first flat-fieldoptical system, which transforms the angular deflection into a parallelshift, and the laser beam pulses of short-wavelength light pass,downstream of the multiplier crystal system through a second flat-fieldoptical system, which transforms the parallel shift back into an angulardeflection.